The activation of phospholipase D (PLD) by external signals is a well- documented fact. We and others have shown that receptor-induced PLD activation is mediated by small G proteins by mechanisms that are relatively specific and distinct for different receptors and cell types. In particular, during the initial funding period of this research project, we have shown that small G proteins of the ARF family are essential for the activation of PLD by insulin and PDGF in HIRcB cells and for the activation of PLD by PDGF and endothelin-1 in the A10 vascular smooth cell line. However, the mechanisms by which the activation of ARF proteins might be regulated by insulin and other extracellular signals remains obscure. This issue is particularly complicated by the existence of many RF proteins (ARF1-ARF6), ARF-GDP exchange factors (ARF-GEF; namely ARNO, cytohesin-1, GRP1, Gea and others) and PLD forms (PLD1 and PLD2) which suggest parallel redundant pathways for the activation of PLD. During the preceding funding period we have started studying the mechanisms by which insulin and other growth factors may regulate the state of activation of ARF. We showed that on ARF-GEF co-immunoprecipitated with the insulin receptor in an insulin-dependent manner. Additional experiments suggest the ARF-GEF known as ARNO associates to the insulin receptor. We propose to study the mechanisms by which the ARF-GEFs ARNO and cytohesin-1 are activated by cell surface receptors and to determine the role that the activation of these GEFs plays in the regulation of PLD by insulin, PDGF and phorbol esters. We propose to do this by a combination of biochemical, molecular and imaging approaches. We will concentrate on the following studies: a) analysis of the role of ARNO and cytohesin-1 on receptor-mediated ARF and PLD activation, by mutational analysis of the Sec7 and pleckstrin-homology domain of the ARF-GEFs and of specific residues of the Switch 1 region of ARF; b) determining the mechanisms of regulation of ARNO and cytohesin-1 by examination of their state of phosphorylation, their interaction with cell surface receptors and their recruitment by phosphoinositides: and c) determining the relative role of PLD1 and PLD2 in receptor-dependent phospholipase D activity.